The diagnostic imaging of cancer using a radioactive nuclide allows a noninvasive early diagnosis of cancer. Technetium-99m (99mTc), a metallic radioisotope (RI), is the most suitable for clinical application among RIs commonly used for diagnostic imaging, since it has a half-life (6 hours) and a γ-ray energy (141 keV) suitable for diagnostic imaging and this nuclide is readily available as a physiology saline solution resulted from a generator system using 99Mo as a parent nuclide. There have been conducted many researches utilizing an antibody or a peptide as a labeling material for the purpose of selectively delivering 99mTc to the tumor, and a liposome is also one of the carrier candidates for use in diagnostic imaging. A liposome is a closed vesicle composed of a lipid bilayer membrane. A liposome draws attention as a capsule type DDS carrier for a medicament such as chemotherapeutic drug, a protein, a nucleic acid, etc. Since liposomes can carry a large amount of radioactivity therein and also be formed target directed by the adjustment of particle size and chemical modification of the membrane surface, applications of 99mTc-labeled liposomes to a high-sensitive diagnostic imaging of solid cancer, sentinel lymph-node and inflammation and infection sites are expected to be useful in nuclear medicine diagnosis as well.
It has been demonstrated that since tumor tissue has increased vascular permeability and lacks in recovery of substances via lymphatic system, macromolecules tend to infiltrate from the blood to the tumor interstitium and accumulate therein. It is also suggested by these properties that liposomes permeated into the tissue interstitium are not taken up into cells and rather stay in the cell interstitium in tumor tissue. Liposomes which circulate through a blood flow, however, are mainly captured by reticuloendothelial tissues such as liver or spleen, and are removed from the blood. Although liposomes wherein a nitrilotriacetic acid (NTA) complex of 67Ga and 111In is encapsulated have been well investigated as well for the purpose of diagnostic imaging of tumors and have exhibited excellent tumor accumulating properties in laboratory animals, they also exhibit high radioactivity accumulation in the liver and spleen, which has been a serious obstacle for practical application thereof. The liposomes incorporated into the liver and spleen are fused with lysosomes in the parenchymal cells, and are then metabolized. Encapsulated complexes of 67Ga and 111In-NTA released in the lysosomes at this time are water-soluble and cannot penetrate the membrane. The complexes decompose due to their low stability, and radioactive nuclides are accumulated in the lysosomes. This is supposed to the cause of the long-lasting radioactivity retention appearing in these tissues.
Accordingly, the present inventors considered that the non-specific retention of radioactivity would be dissipated by conferring properties to move from the lysosome into the blood and to be quickly excreted into the urine on the RI complexes released after liposomes are incorporated into the lysosomes in the cell and metabolized and released therefrom. 99mTc-ethylendicysteine (99mTc-CD) (FIG. 1) has been selected as a complex having such properties. 99mTc-CD has two molecules of free carboxylic acid and a stable pentavalent neutral complex structure. It is reported that 99mTc-CD is excreted into the urine in a stable chemical form through the organic anion transporter in the kidney as in the case of para-aminohippuric acid. According to the researches of the present inventors, it has been demonstrated that when 186Re-CD, a CD complex of rhenium-186 (186Re) which is a long half-life metallic radioactive nuclide of the same family as 99mTc is encapsulated in the liposome, radioactivity accumulated in the liver and spleen can be promptly excreted into urine as 186Re-CD, and radioactivity retained in these internal organs can be significantly reduced. These results suggest that the radioactivity retained in the liver or spleen can be dissipated also with the use of liposomes wherein 99mTc-CD complexes are encapsulated.
However, when a direct encapsulation approach was taken at the time of preparing 186Re-CD, where 186Re-CD complex is added to lipid as it is and then subjected to swelling, encapsulating efficiency is extremely low as 3.2%. Accordingly, it is necessary to establish a method of encapsulating 186Re-CD or 99mTc into liposomes. Moreover, in case of clinical use, direct encapsulation approach requires liposomes to be prepared just before use. It is practically desired, however, that the liposomes prepared beforehand are labeled when clinically used. A technique has been reported as an efficient and simple encapsulating method for 67Ga and 111In, in which 67Ga and 111In-oxine complex having a high lipid solubility and substitution activity is prepared and is incubated with NTA-encapsulated liposomes so that the complex penetrates the liposome membrane and causes a ligand exchange reaction in the liposomes thereby retaining these nuclides as water-soluble chelate 67Ga and 111In-NTA within the liposomes (FIG. 2). When this encapsulating method called ligand exchange reaction is used, the encapsulation efficiency of 67Ga or 111In reaches to about 90%. Another technique for obtaining 99mTc-labeled liposomes in high radiochemical yield comprises incubating 99mTc-hexamethyl propyleneamine oxime (99mTc-HMPAO) which is a lipophilic complex with glutathione-encapsulated liposomes, thereby reductively converting the 99mTc-HMPAO within the liposome into a water-soluble decomposed product. The encapsulating efficiency by this technique is about 60% to 90%. Recently, RI labeled liposomes used in nuclear medicine are most commonly prepared by this encapsulating method. This method, however, give rise to problems of radioactivity retention in the liver and spleen as mentioned above for each of the 67Ga, 111In and 99mTc labeled liposomes.